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. 2000 Jul 1;349(Pt 1):135–140. doi: 10.1042/0264-6021:3490135

The short prodomain influences caspase-3 activation in HeLa cells.

T Meergans 1, A K Hildebrandt 1, D Horak 1, C Haenisch 1, A Wendel 1
PMCID: PMC1221130  PMID: 10861221

Abstract

Proteolytic activation of caspases is a key step in the process of apoptosis. According to their primary structure, caspases can be divided into a group with a long prodomain and a group with a short prodomain. Whereas long prodomains play a role in autocatalytic processing, little is known about the function of the short prodomain, for example the prodomain of caspase-3. We constructed caspase-3 variants lacking the prodomain and overexpressed these in HeLa and yeast cells. We found that removal of the caspase-3 prodomain resulted in spontaneous proteolytic activation of the protein when expressed in HeLa cells. This processing was only partially autocatalytic, as demonstrated by a catalytically inactive caspase-3 mutant. Co-expression of the anti-apoptotic protein XIAP (X-chromosome-linked inhibitor of apoptosis protein) completely blocked the observed spontaneous activation, which excluded a direct involvement of caspase-8. Our findings indicate that the short prodomain of caspase-3 serves as a silencing component in mammalian cells by retaining this executioner caspase in an inactive state.

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Selected References

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  1. Alnemri E. S., Livingston D. J., Nicholson D. W., Salvesen G., Thornberry N. A., Wong W. W., Yuan J. Human ICE/CED-3 protease nomenclature. Cell. 1996 Oct 18;87(2):171–171. doi: 10.1016/s0092-8674(00)81334-3. [DOI] [PubMed] [Google Scholar]
  2. Boldin M. P., Goncharov T. M., Goltsev Y. V., Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996 Jun 14;85(6):803–815. doi: 10.1016/s0092-8674(00)81265-9. [DOI] [PubMed] [Google Scholar]
  3. Butt A. J., Harvey N. L., Parasivam G., Kumar S. Dimerization and autoprocessing of the Nedd2 (caspase-2) precursor requires both the prodomain and the carboxyl-terminal regions. J Biol Chem. 1998 Mar 20;273(12):6763–6768. doi: 10.1074/jbc.273.12.6763. [DOI] [PubMed] [Google Scholar]
  4. Casciola-Rosen L., Nicholson D. W., Chong T., Rowan K. R., Thornberry N. A., Miller D. K., Rosen A. Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death. J Exp Med. 1996 May 1;183(5):1957–1964. doi: 10.1084/jem.183.5.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chinnaiyan A. M., O'Rourke K., Tewari M., Dixit V. M. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell. 1995 May 19;81(4):505–512. doi: 10.1016/0092-8674(95)90071-3. [DOI] [PubMed] [Google Scholar]
  6. Cohen G. M. Caspases: the executioners of apoptosis. Biochem J. 1997 Aug 15;326(Pt 1):1–16. doi: 10.1042/bj3260001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Colussi P. A., Harvey N. L., Shearwin-Whyatt L. M., Kumar S. Conversion of procaspase-3 to an autoactivating caspase by fusion to the caspase-2 prodomain. J Biol Chem. 1998 Oct 9;273(41):26566–26570. doi: 10.1074/jbc.273.41.26566. [DOI] [PubMed] [Google Scholar]
  8. Cryns V. L., Bergeron L., Zhu H., Li H., Yuan J. Specific cleavage of alpha-fodrin during Fas- and tumor necrosis factor-induced apoptosis is mediated by an interleukin-1beta-converting enzyme/Ced-3 protease distinct from the poly(ADP-ribose) polymerase protease. J Biol Chem. 1996 Dec 6;271(49):31277–31282. doi: 10.1074/jbc.271.49.31277. [DOI] [PubMed] [Google Scholar]
  9. Deveraux Q. L., Roy N., Stennicke H. R., Van Arsdale T., Zhou Q., Srinivasula S. M., Alnemri E. S., Salvesen G. S., Reed J. C. IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J. 1998 Apr 15;17(8):2215–2223. doi: 10.1093/emboj/17.8.2215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deveraux Q. L., Takahashi R., Salvesen G. S., Reed J. C. X-linked IAP is a direct inhibitor of cell-death proteases. Nature. 1997 Jul 17;388(6639):300–304. doi: 10.1038/40901. [DOI] [PubMed] [Google Scholar]
  11. Dorstyn L., Kinoshita M., Kumar S. Caspases in cell death. Results Probl Cell Differ. 1998;24:1–24. doi: 10.1007/978-3-540-69185-3_1. [DOI] [PubMed] [Google Scholar]
  12. Green D. R. Apoptotic pathways: the roads to ruin. Cell. 1998 Sep 18;94(6):695–698. doi: 10.1016/s0092-8674(00)81728-6. [DOI] [PubMed] [Google Scholar]
  13. Han Z., Hendrickson E. A., Bremner T. A., Wyche J. H. A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro. J Biol Chem. 1997 May 16;272(20):13432–13436. doi: 10.1074/jbc.272.20.13432. [DOI] [PubMed] [Google Scholar]
  14. Hu Y., Ding L., Spencer D. M., Núez G. WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation. J Biol Chem. 1998 Dec 11;273(50):33489–33494. doi: 10.1074/jbc.273.50.33489. [DOI] [PubMed] [Google Scholar]
  15. Kothakota S., Azuma T., Reinhard C., Klippel A., Tang J., Chu K., McGarry T. J., Kirschner M. W., Koths K., Kwiatkowski D. J. Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science. 1997 Oct 10;278(5336):294–298. doi: 10.1126/science.278.5336.294. [DOI] [PubMed] [Google Scholar]
  16. Li P., Nijhawan D., Budihardjo I., Srinivasula S. M., Ahmad M., Alnemri E. S., Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997 Nov 14;91(4):479–489. doi: 10.1016/s0092-8674(00)80434-1. [DOI] [PubMed] [Google Scholar]
  17. Martin D. A., Siegel R. M., Zheng L., Lenardo M. J. Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACHalpha1) death signal. J Biol Chem. 1998 Feb 20;273(8):4345–4349. doi: 10.1074/jbc.273.8.4345. [DOI] [PubMed] [Google Scholar]
  18. Medema J. P., Scaffidi C., Kischkel F. C., Shevchenko A., Mann M., Krammer P. H., Peter M. E. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 1997 May 15;16(10):2794–2804. doi: 10.1093/emboj/16.10.2794. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meergans T., Albig W., Doenecke D. Conserved sequence elements in human main type-H1 histone gene promoters: their role in H1 gene expression. Eur J Biochem. 1998 Sep 1;256(2):436–446. doi: 10.1046/j.1432-1327.1998.2560436.x. [DOI] [PubMed] [Google Scholar]
  20. Muzio M., Chinnaiyan A. M., Kischkel F. C., O'Rourke K., Shevchenko A., Ni J., Scaffidi C., Bretz J. D., Zhang M., Gentz R. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell. 1996 Jun 14;85(6):817–827. doi: 10.1016/s0092-8674(00)81266-0. [DOI] [PubMed] [Google Scholar]
  21. Muzio M., Stockwell B. R., Stennicke H. R., Salvesen G. S., Dixit V. M. An induced proximity model for caspase-8 activation. J Biol Chem. 1998 Jan 30;273(5):2926–2930. doi: 10.1074/jbc.273.5.2926. [DOI] [PubMed] [Google Scholar]
  22. Nicholson D. W., Ali A., Thornberry N. A., Vaillancourt J. P., Ding C. K., Gallant M., Gareau Y., Griffin P. R., Labelle M., Lazebnik Y. A. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995 Jul 6;376(6535):37–43. doi: 10.1038/376037a0. [DOI] [PubMed] [Google Scholar]
  23. Nicholson D. W., Thornberry N. A. Caspases: killer proteases. Trends Biochem Sci. 1997 Aug;22(8):299–306. doi: 10.1016/s0968-0004(97)01085-2. [DOI] [PubMed] [Google Scholar]
  24. Rotonda J., Nicholson D. W., Fazil K. M., Gallant M., Gareau Y., Labelle M., Peterson E. P., Rasper D. M., Ruel R., Vaillancourt J. P. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis. Nat Struct Biol. 1996 Jul;3(7):619–625. doi: 10.1038/nsb0796-619. [DOI] [PubMed] [Google Scholar]
  25. Roy N., Deveraux Q. L., Takahashi R., Salvesen G. S., Reed J. C. The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J. 1997 Dec 1;16(23):6914–6925. doi: 10.1093/emboj/16.23.6914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Saleh A., Srinivasula S. M., Acharya S., Fishel R., Alnemri E. S. Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J Biol Chem. 1999 Jun 18;274(25):17941–17945. doi: 10.1074/jbc.274.25.17941. [DOI] [PubMed] [Google Scholar]
  27. Salvesen G. S., Dixit V. M. Caspases: intracellular signaling by proteolysis. Cell. 1997 Nov 14;91(4):443–446. doi: 10.1016/s0092-8674(00)80430-4. [DOI] [PubMed] [Google Scholar]
  28. Scaffidi C., Fulda S., Srinivasan A., Friesen C., Li F., Tomaselli K. J., Debatin K. M., Krammer P. H., Peter M. E. Two CD95 (APO-1/Fas) signaling pathways. EMBO J. 1998 Mar 16;17(6):1675–1687. doi: 10.1093/emboj/17.6.1675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Slee E. A., Harte M. T., Kluck R. M., Wolf B. B., Casiano C. A., Newmeyer D. D., Wang H. G., Reed J. C., Nicholson D. W., Alnemri E. S. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol. 1999 Jan 25;144(2):281–292. doi: 10.1083/jcb.144.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Srinivasula S. M., Ahmad M., Fernandes-Alnemri T., Alnemri E. S. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol Cell. 1998 Jun;1(7):949–957. doi: 10.1016/s1097-2765(00)80095-7. [DOI] [PubMed] [Google Scholar]
  31. Srinivasula S. M., Ahmad M., MacFarlane M., Luo Z., Huang Z., Fernandes-Alnemri T., Alnemri E. S. Generation of constitutively active recombinant caspases-3 and -6 by rearrangement of their subunits. J Biol Chem. 1998 Apr 24;273(17):10107–10111. doi: 10.1074/jbc.273.17.10107. [DOI] [PubMed] [Google Scholar]
  32. Stennicke H. R., Deveraux Q. L., Humke E. W., Reed J. C., Dixit V. M., Salvesen G. S. Caspase-9 can be activated without proteolytic processing. J Biol Chem. 1999 Mar 26;274(13):8359–8362. doi: 10.1074/jbc.274.13.8359. [DOI] [PubMed] [Google Scholar]
  33. Stennicke H. R., Jürgensmeier J. M., Shin H., Deveraux Q., Wolf B. B., Yang X., Zhou Q., Ellerby H. M., Ellerby L. M., Bredesen D. Pro-caspase-3 is a major physiologic target of caspase-8. J Biol Chem. 1998 Oct 16;273(42):27084–27090. doi: 10.1074/jbc.273.42.27084. [DOI] [PubMed] [Google Scholar]
  34. Talanian R. V., Quinlan C., Trautz S., Hackett M. C., Mankovich J. A., Banach D., Ghayur T., Brady K. D., Wong W. W. Substrate specificities of caspase family proteases. J Biol Chem. 1997 Apr 11;272(15):9677–9682. doi: 10.1074/jbc.272.15.9677. [DOI] [PubMed] [Google Scholar]
  35. Tewari M., Quan L. T., O'Rourke K., Desnoyers S., Zeng Z., Beidler D. R., Poirier G. G., Salvesen G. S., Dixit V. M. Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell. 1995 Jun 2;81(5):801–809. doi: 10.1016/0092-8674(95)90541-3. [DOI] [PubMed] [Google Scholar]
  36. Thornberry N. A. Interleukin-1 beta converting enzyme. Methods Enzymol. 1994;244:615–631. doi: 10.1016/0076-6879(94)44045-x. [DOI] [PubMed] [Google Scholar]
  37. Thornberry N. A., Rano T. A., Peterson E. P., Rasper D. M., Timkey T., Garcia-Calvo M., Houtzager V. M., Nordstrom P. A., Roy S., Vaillancourt J. P. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem. 1997 Jul 18;272(29):17907–17911. doi: 10.1074/jbc.272.29.17907. [DOI] [PubMed] [Google Scholar]
  38. Yang X., Chang H. Y., Baltimore D. Autoproteolytic activation of pro-caspases by oligomerization. Mol Cell. 1998 Jan;1(2):319–325. doi: 10.1016/s1097-2765(00)80032-5. [DOI] [PubMed] [Google Scholar]
  39. Zou H., Henzel W. J., Liu X., Lutschg A., Wang X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell. 1997 Aug 8;90(3):405–413. doi: 10.1016/s0092-8674(00)80501-2. [DOI] [PubMed] [Google Scholar]

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